Combining elements of sequence-specificity and catalytic chemistry is the central problem in developing chemotherapeutics which target specific nucleic acid sequences and structures. Restriction enzymes, of which EcoRI endonuclease is most representative, are the simplest biological agents of site-specific nucleic acid chemistry, yet many mechanistic aspects of this hydrolytic activity remain undefined. This project employs a unique combination of techniques to: i) Identify the protein and substrate ligands of the required metal ion (MG(II)) and define the importance of these interactions in the reaction. These goals will be accomplished with site-directed mutagenesis, metal ion substitution, substrate analogs, inert transition metal complexes, and magnetic resonance spectroscopy. ii) Characterize the hydration of the required metal ion by spectroscopic methods and probe the importance of proton transfer in DNA hydrolysis using kinetic isotope effects. iii) Probe the role of conformational changes in mediating EcoRI endonuclease substrate specificity and star activity. EcoRI endonuclease labelled with 3-fluoro--tyrosine will be prepared and characterized. 19/F NMR spectroscopy will be applied to this enzyme derivative to determine if substrate specificity and star activity have a physical basis in conformation. Information obtained from these experiments will identify the relationships between the enzyme structure, the required metal ion, and the substrate as well as clarify working models of EcoRI activity. Understanding the structural and functional aspects of this natural form of specific, hydrolytic catalysis is a viable strategy to the eventual design of sequence-specific scissors which can target specific genes linked to cancer and AIDS.